Experiments have been carried out to demonstrate that the technique of continuous internal and external perfusion of a single barnacle giant muscle fiber is suitable for 3-0-methyl-D-glucose (3-0-MG) transport studies. In the absence of experimental perturbation, both membrane potential and efflux of C14 labeled 3-0-MG remain constant for several hours. It was found that C14 3-0-MG efflux is strongly inhibited by phlorizin. It is also decreased by the addition of large concentrations of unlabeled 3-0-MG or unlabeled D-glucose to internal perfusates containing small concentrations of C14 3-0-MG. It is concluded that the majority of C14 3-0-MG efflux is due to a specific transport system in the membrane, and not due to leakage. The large decrease in C14 3-0-MG efflux produced by increased internal D-glucose concentration indicates that both C14 3-0-MG and D-gucose are transported by the same carrier. The main objectives of the research proposed here are to use this technique to 1) obtain a kinetic characterization of C14 3-0-MG efflux and 2) identify factors that effect C14 3-0-MG efflux. Attention will be focused on testing substances which are either known or thought to effect glucose transport in insulin-sensitive tissues in higher animals: Ca ions, Mg ions, Na ion, K ion, insulin, ATP, cyclic nucleotides, membrane lipids, and membrane potential. To test the effect of a given substance, the concentration of the substance in the appropriate fluid (internal or external) will be changed, and subsequent changes in C14 3-0-MG efflux will be measured. To test the effect of membrane potential, the membrane will be either hyperpolarized or depolarized by sending current. Despite large amounts of research, the factors exerting everyday, direct physiological control over glucose transport in insulin-sensitive cells remain largely unidentified. Because this technique provides continuous control over the composition of both the internal and external fluids, it shows considerable promise for leading to the identity of such factors.